Project Summary/Abstract A long-held tenet of molecular pharmacology is that the plasma membrane is the sole site of action of external cues, such as peptide hormones and biogenic amines, which cannot cross this barrier. The centerpiece of this proposal emerges from my discoveries that a set of receptors long considered to only signal from the plasma membrane, also signal from subcellular membrane compartments. This compartmentalization of signaling challenges some of the basic paradigms of signaling regulation. While the existing data already shows that compartmentalized signaling is a general feature of many membrane receptors, the focus of this proposal is on ?-adrenergic receptors (?ARs) class of G Protein Coupled Receptors (GPCRs). ?ARs control the strength and frequency of cardiac contraction and disturbances in ?AR signaling underlie hypertension and heart failure. ?ARs have served as a prototypical receptor whose sites of action are limited to plasma membrane and removal of the receptors from the plasma membrane has been generally viewed as the mechanism by which signaling is terminated. Combining sophisticated imaging platforms with conformational biosensor that I developed, I directly probed activation of ?AR and its cognate Gs protein. I discovered that active ?AR-G?s complex is not restricted to the plasma membrane but is also actively signals at subcellular membrane compartments such as endosomes and the Golgi. I further showed that impermeable hormones such as epinephrine/norepinephrine can reach the Golgi membranes through a mechanism facilitated by an organic cation transporter (OCT3). These findings have uncovered an entirely new regulatory component to ?AR signaling, namely intracellular, compartmentalized signaling. My long-term goal is to broadly understand the functional consequence of compartmentalized signaling as a way of integrating cell biology and signaling with physiology and pathophysiology. To facilitate this process, my laboratory will focus on ?AR signaling from subcellular membrane compartment in regulating cardiac functions. In this proposal we plan to: 1) Elucidate the molecular and cellular consequences of ?AR compartmentalized signaling in mouse-derived cardiomyocytes, 2) Elucidate the mechanism of activation and inactivation of ?ARs at different internal membrane compartments 3) Elucidate the role of compartmentalized signaling in regulating cardiac outputs in zebrafish. We have developed new tools, using nanobodies, to disrupt receptor/G protein coupling at specific membrane locations and are combining them with an optogenetic approach to inhibit compartmentalized ?ARs signaling in a dose dependent and reversible manner. Currently, GPCRs are among the most heavily sought after drug targets. Most of these efforts fail to consider that receptor manipulation at different subcellular compartments can cause vastly different outcomes, a notion that is already hinted at by our preliminary studies. Thus, our efforts at understanding compartmentalized signaling has the potential of transforming the strategies that are used for developing effective small molecule therapeutics that link to GPCR signaling.